胰岛素调节人胰腺癌细胞ASPC-1中HIF-1α的表达研究

目的 探讨胰岛素对胰腺癌细胞株ASPC-1中缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响.方法 将ASPC-1细胞分为5组:常氧组,常氧加胰岛素组,缺氧组,同一缺氧时间不同胰岛素浓度组以及相同胰岛素浓度不同缺氧时间组,实时定量PCR检测HIF-1α基因表达,免疫细胞化学与Western blot检测HIF-1α蛋白表达水平的变化.Transwell实验检测加入胰岛素后肿瘤细胞侵袭能力的变化.结果 常氧下ASPC-1细胞即有一定水平HIF-1α表达,加入胰岛素刺激后HIF-1α蛋白表达随着胰岛素浓度的升高和作用时间的延长而逐渐升高,与对照组相比差异有统计学意义(P＜0.05);缺氧条件下HIF-1α表达较常氧时升高(P＜0.05);相同缺氧时间使用不同浓度胰岛素刺激时,高浓度胰岛素刺激下HIF-1α的表达较单纯缺氧更高(P＜0.05),低浓度胰岛素对HIF-1α无明显作用(P＞0.05);使用相同胰岛素处理后再进行缺氧处理,随着时间延长HIF-1α的表达先升高后下降,但各处理组较对照组差异均有统计学意义(P＜0.05).Transwell实验显示胰岛素可增强胰腺癌细胞的侵袭能力(P＜0.05).结论 胰岛素可上调胰腺癌细胞ASPC-1中HIF-1蛋白表达,并且这种作用具有一定的剂量依赖性和时间依赖性.胰岛素还可以增加ASPC-1的侵袭能力.

Abstract：

Objective To investigate the effect of insulin on the expression of hypoxia-inducible factor-1α in human pancreatic cancer cell line ASPC-1. Methods We divided ASPC-1 cells into five groups: normoxia; normoxia stimulated with insulin; hypoxia; hypoxia pretreated with different concentration of insulin; hypoxia of different time points pretreated with same concentration of insulin. Real-time PCR was used to test the expression of HIF-1α mRNA. Immunohistochemistry was used to examine the expression of HIF-1α in ASPC-1 of insulin treated cancer cells. Western blot was used to determine the expression of HIF-1 α protein in those cells. Transwell was used to test whether insulin could enhance the invasion ability of ASPC-1 pancreatic cancer cells. Results Insulin promotes HIF-1α protein expression. ASPC-1 cells expressed low levels of HIF-1α protein under normoxic condition. After stimulated with insulin, the expression of HIF-1 α protein significantly increased (P ＜ 0. 05 ). After treated with hypoxia, the expression of HIF-1α protein also increased(P ＜ 0. 05 ). Low concentrations of insulin didn't increase the expression of HIF-1α under hypoxic environment ( P ＞ 0. 05 ), while high concentration of insulin could increase its expression(P ＜ 0. 05). When ASPC-1 cells pretreated with insulin suffered from hypoxia, the expression of HIF-1α first increased then decreased moderately( P ＜0. 05). Insulin could enhance the invasion ability of pancreatic cancer cells( P ＜ 0. 05 ). Conclusions Insulin mediates the expression of HIF-1α protein in human pancreatic cancer ASPC-1 cells with the characteristics of dose and time dependency. Insulin could enhance the invasion ability of ASPC-1 cells.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

RhoA基因沉默对肝癌HepG2细胞增殖和迁移能力的影响

Objective To construct a RhoA-siRNA expression vector and determine its role on the malig-nant behavior of HepG2 cells.Methods A RhoA-siRNA DNA fragment was synthesized and cloned into the expression vector of pGenesil-1.The constructed Rhon-siRNA DNA plasmid was stably transfected into HerG2 cells by lipofectamine,and then HepG2 cells were divided into the HepG2/RhoA-siRNA group (HepG2 cells were transfected with pGenesil-1-RhoA-siRNA),HepG2/control group(HepG2 cells were transfected with control plasmid) and HepG2 group (without plasmid transfection).The inbibitory effect of RhoA-siRNA on RhoA protein expression was shown by Western blot.The proliferation,migration,growth potentiality and cell cycle of transfected HepG2 cells were evaluated by MTT assay,wounded healing,the plate cloning formation test and flow cytometry,respectively.All data were analyzed by one-way analysis of variance (ANOVA) and chi-square test.Results The expression of RhoA protein in the HepG2/RhoA-siRNA group was,significantly decreased compared with that in the other two groups (F=178.19,P<0.05).Scratched cells were healed within 48 hours in the HepG2/control group and HepG2 group,but not in the HepG2/RhoA-siRNA group.The clone formation rates in the HepG2/RhoA-siRNA group,HepG2 group and HepG2/control group were 39%±3%,67%±5%and 70%±6%,respectively,with a significant difference among the three groups(χ2=33.34,38.69,P<0.05).Flow cytometry showed that the number of cells transfected with RhoA-siRNA was highest in the G0/G1 phase and lowest in the S phase(F=70.46,76.57.P<0.05).Conclusion The RhoA-siRNA expression vector can effectively suppress the proliferation and migration of HepG2 cells,which may provide a novel gene therapy for hepatocellular carcinoma.     

缺氧诱导因子-1α反义寡核苷酸体外对胶质瘤U87细胞增殖和侵袭的影响

目的 观察缺氧诱导因子-1α(HIF-1α)反义寡核苷酸体外对人胶质瘤U87细胞增殖、凋亡和侵袭的影响.方法 人工合成的HIF-1α反义短发夹经阳离子脂质体包裹后瞬时转染人胶质瘤细胞株U87,采用Western blot法检测转染后胶质瘤细胞HIF-1α蛋白表达,证实转染成功,再采用噻唑蓝(MTT)比色法和Transwell小室体外侵袭实验检测转染后对U87细胞增殖体外侵袭能力的影响,流式细胞仪检测细胞凋亡.结果 转染靶向HIF-1α的短链发夹RNA的胶质瘤细胞,HIF-1α蛋白表达较空白细胞组明显降低,MTT法检测转染组、空载体组和空细胞组24 h细胞的增殖率分别为5.46%、21.25%、22.32%,体外侵袭性实验表明转染组、空载体组和空细胞组细胞12 h侵袭ECM的细胞数分别为(22±4)、(124±3)、(122±6);流式细胞仪检测表明转染组、空载体组和空细胞组细胞凋亡率分别为(53.35±2.80)%、(12.02±1.60)%、(10.19±3.15)%,转染组与空白细胞组及空载体组比较差异均有统计学意义(P＜0.05).结论 封闭HIF-1α蛋白的表达,可以抑制人胶质瘤U87细胞增殖和侵袭能力,促进细胞凋亡.

Abstract：

Objective To investigate the effects of antisense oligodeoxynucleotides (ODNs) targeting hypoxia inducible factor-1α (HIF-1α) on the apeptosis, proliferation and invasion of U87 glioma cell line. Methods Antisense ODNs were constructed and transfected into U87 cells by Dosper liposomal reagent. The HIF-1α gene expression was detected by Western blotting, the cell proliferative index was determined by methyl thiazol tetrazolium (MTT) assay, the cells cycle and apoptosis of the cells were examined by flow cytometry and the changes of the U87 cells invasive ability were measured by Transwell chamber.Results The protein expression of HIF-1α in U87 cells was down-regulated by HIF-1α ASONDN. The cell proliferative index in transfected group, empty vector group and control group was 5.46％, 21.25％and 22. 32％ respectively. Transwell chamber assay showed that the cell number in transfected group,empty vector group and control group was (22 ±4), ( 124 ±3) and ( 122 ±6) respectively; and the apoptosis rate was (53. 35 ± 2. 80) ％, ( 12.02 ± 1.60 )％, ( 10. 19 ± 3. 15 )％ respectively, and there was significant difference between transfected group and other groups ( P ＜ 0. 05 ). Conclusion Silencing the expression of HIF-1α protein can inhibit proliferation and invasion, and promote apoptosis of human glioma U87 cells. HIF-1α is expected to become a target for cancer therapy.     

HIF-1α、P-gp在胰腺癌中的表达及其临床病理学的意义

目的 检测HIF-1α、P-gp蛋白在胰腺癌组织中的表达,探讨它们的临床病理学意义及其相互之间的相关性.方法 应用免疫组织化学法检测74例胰腺癌组织,10例正常胰腺组织中HIF-1α、P-gp蛋白的表达.结果 HIF-1α、P-gp蛋白在胰腺癌组织中的阳性表达率分别为75.7%和86.5%,而正常胰腺组织中均呈阴性表达(P＜0.05).胰腺癌组织中HIF-1α和P-gp蛋白的表达呈显著正相关(r=0.304,P=0.009).胰腺癌组织中的HIF-1α蛋白表达与淋巴结转移、TNM分期呈正相关(P＜0.05);胰腺癌组织中的P-gp蛋白表达与淋巴结转移呈正相关,P-gp高表达与年龄大于60岁有关(P＜0.05).HIF-1α和P-gp蛋白高表达者的中位生存期明显短于低表达者(P＜0.05).结论 胰腺癌组织中HIF-1α、P-gp蛋白的表达上调,HIF-1α和P-gp呈正相关.胰腺癌组织中HIF-1α和P-gp蛋白的表达与淋巴结转移呈正相关.胰腺癌组织中HIF-1α和P-gp蛋白的高表达与预后差有关.

Abstract：

Objective To investigate the expression of HIF-1α and P-gp protein in pancreatic carcinoma and determine their clinicopathological significance and the correlation between the expression of HIF-1α, P-gp and the clinical prognosis. Method In samples from 74 cases of pancreatic carcinoma and 10 healthy individuals, the expression of HIF-1α and P-gp were detected by immunohistochemical method. Results The positive expression rate of HIF-1α and P-gp was 75.7% and 86.5%,respectively, which were remarkably higher than that in normal pancreatic tissue (P＜0.05). There was a positive correlation between the expression of HIF-1α and that of P-gp. The aberrant expression of HIF-1α and P-gp was associated with lymph node metastasis but not the location, size, clinical stages and nerve invasion of the tumor. Patients with high intensity of HIF-1α and P-gp expression showed a significantly lower median survival time than those with low intensity expression.Conclusions The expression of HIF-1α and P-gp is up-regulated in pancreatic carcinoma and there is a positive correlation between them. The expression of HIF-1α and P-gp might be related to the lymph node metastasis and poor prognosis.     

鼠双微体2蛋白参与醛固酮诱导的系膜细胞增殖

Objective To investigate whether and how murine double minute 2(MDM2) was involved in aldosterone (ALD)-induced human mesangial cells line (HMCLs) proliferation. Methods RT-PCR and immunofluorescence were used to confirm the expression of MDM2 in HMCLs. Western blotting was used to estimate the relationship between ALD dose and MDM2 expression. Spironolactone, a mineralocorticoid receptor (MR) blocker, was used to estimate the role of MR on the up-regulation of MDM2 induced by ALD. Cycloheximide (CHX), a protein synthesis inhibitor, was used to estimate whether the rapid nongenomic mechanism was involved in the upregulation. To confirm the relationship among ALD, MDM2 expression and proliferation of HMCLs, small interference RNA of MDM2 was applied. Results Both MR and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) mRNAs were detected in HMCLs. MDM2 protein expression was also detected in both the nucleus and the cytoplasm. ALD significantly stimulated MDM2 expression, which implied that MDM2 was a novel mineralocorticoid-responsive gene in HMCLs. MR was involved in this process as spironolactone did not promote the expression of MDM2 mRNA or protein. ALD with CHX did not increase the expression of MDM2 protein, which indicated it was not directly regulated by the rapid nongenomic mechanisms. MDM2 protein was decreased by using the transfection of MDM2 siRNA and ALD did not promote the cell proliferation of HMCLs under the same conditions. All of which implied that MDM2 participated in ALQ-induced HMCLs proliferation. Conclusions MDM2 is a novel mineralocorticoid-responsive gene in HMCLs. MR is involved in ALD-induced MDM2 expression which is inhibited by spironolactone. The increased expression of MDM2 protein induced by ALD is not directly regulated by the rapid nongenomic mechanisms. MDM2 participates in ALD induced HMCLs proliferation.     

鼠双微体2蛋白参与醛固酮诱导的系膜细胞增殖

Objective To investigate whether and how murine double minute 2(MDM2) was involved in aldosterone (ALD)-induced human mesangial cells line (HMCLs) proliferation. Methods RT-PCR and immunofluorescence were used to confirm the expression of MDM2 in HMCLs. Western blotting was used to estimate the relationship between ALD dose and MDM2 expression. Spironolactone, a mineralocorticoid receptor (MR) blocker, was used to estimate the role of MR on the up-regulation of MDM2 induced by ALD. Cycloheximide (CHX), a protein synthesis inhibitor, was used to estimate whether the rapid nongenomic mechanism was involved in the upregulation. To confirm the relationship among ALD, MDM2 expression and proliferation of HMCLs, small interference RNA of MDM2 was applied. Results Both MR and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) mRNAs were detected in HMCLs. MDM2 protein expression was also detected in both the nucleus and the cytoplasm. ALD significantly stimulated MDM2 expression, which implied that MDM2 was a novel mineralocorticoid-responsive gene in HMCLs. MR was involved in this process as spironolactone did not promote the expression of MDM2 mRNA or protein. ALD with CHX did not increase the expression of MDM2 protein, which indicated it was not directly regulated by the rapid nongenomic mechanisms. MDM2 protein was decreased by using the transfection of MDM2 siRNA and ALD did not promote the cell proliferation of HMCLs under the same conditions. All of which implied that MDM2 participated in ALQ-induced HMCLs proliferation. Conclusions MDM2 is a novel mineralocorticoid-responsive gene in HMCLs. MR is involved in ALD-induced MDM2 expression which is inhibited by spironolactone. The increased expression of MDM2 protein induced by ALD is not directly regulated by the rapid nongenomic mechanisms. MDM2 participates in ALD induced HMCLs proliferation.     

5α-reductase type 2 gene expression in human testis, epididymis and vas deferens

To study the expression pattern of 5α-reductase type 2 gene in human male reproductive organs. Methods: The expression level of 5ct-reductase type 2 gene in human testis, epididymis and vas deferens tissues was determined by in situ hybridization using a digoxin-labeled 5α-reductase type 2 cRNA probe. Results: The brown granules of hybridizing signals distributed in the cytoplasm of the Sertoli and Leydig cells of the testis, the principle cells of epididymis and the epithelial cells of vas deferens, but there was no positive signal in the nuclei of these cells. No positive signal was observed in the germ cells, basement of the testis, interstium of the epididymis and basement and the smooth muscle cells of vas deferens. Conclusion: This study confirmed that the 5a-reductase type 2 gene expressed in the Sertoli and Leydig cells of the testis and the principle cells of the epididymis. The expression pattem of the gene in these cells in the human was similar to that in the rat and monkey. The presence o     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

HCCR-2反义核酸对肝癌HepG2细胞的作用

Objective To investigate the effects of antisense recombinant euraryotic expression vector of HCCR-2 on the proliferation and apoptosis of HepG2. Methods The antisense recombinant eukaryotic expression vector of HCCR-2 was constructed. The vector was stably transfected to the HepG2 cells, and positive clones were selected by G418 (antiseuse vector group), pIRES2-EGFP vector was transfected into the HepG2 cells in the same way (pIRES2-EGFP group). The conditions of the nontransfected HepG2 cells were used as control (HepG2 group). Changes in cell growth curve, cell cycle, cell apoptosis and morphology of HepG2 cells after the transfec-tion were detected by MTT method, flow cytometry and transmission electron microscopy, respectively. All the data were analyzed by one-way ANOVA and chi-square test. Results The expression level of HCCR-2 mRNA was down-regulated to 0.39±0.04 in antisense vector group, and the expression level of HCCR-2 mRNA in pIRES2-EGFP group and HepG2 group were 0.62±0.06 and 0.72±0.03, respectively, with significant difference among the 3 groups (F=43.701, P<0.05). The apoptotic rate of HepG2 cells in antisense vector group, pIRES2-EGFP grop and HepG2 group were 13.30%, 2.51% and 2.07%, respectively, with significant difference among the 3 group (χ2=6.793, 8.721, P<0.05). The growth of HepG2 cells in antisense vector group was retarded, and was blocked in G0/G1 stage. Conclusions The HCCR-2 antisense recombinant eukaryotic expression vector can inhibit the mRNA expression of HCCR-2 and promote the apoptosis of cells. HCCR-2 may be involved in cell regulation and the proliferation of hepatocellular carcinoma cells.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.     

Expression and significance of HIF-1α and ET-1 in gastrointestinal stromal tumors

Objective To explore the expression and the value of HIF-1α,and ET-1 in judging the prognosis of gastrointestinal stromal tumors (GISTs). Methods The expression of HIF-1α, and ET-1 protein was examined in 76 GISTs by immunohistochemistry S -P methods. Results There was a positive correlation between the expression of HIF-1 α and ET-1 ( P < 0.05 ). The positive expression rate of HIF-1 α and ET-1 was 73.68% (50/76) ,and 65.79% (50/76) respectively,which was related with histologicial grade, tumor diameter, infiltration and metastasis, nuclear division rating of GISTs ( P < 0.05 ), but had no relationship with patients' age, gender, initial position of the tumor ( P > 0.05 ). There was statistically sig-nificant difference in the expression of HIF-1 α and ET-1 in the following groups:among the three classes of very low-risk and low-risk, middle-risk, high-risk, bewteen the diameter < 2 cm and > 5 cm (P < 0.05). The more malignant degree and larger diameter, the more highly positive expression rate ( P < 0.05 ). The positive expression in the groups with infiltration and metastasis, and nuclear division ≥5/50 HP was sig-nificantly higher than the groups without infiltration and metastasis, and nuclear division < 5/50 HP (P < 0.05). Conclusion The expression of HIF-1α had a significant correlation with ET-1. HIF-1α,and ET-1 expression was closely related with the prognosis of GISTs,and can serve as important predictors for survival.